bafilomycin-a1 and Triple-Negative-Breast-Neoplasms

Epidermal growth factor receptor (EGFR) is over-expressed in about 50% of Triple negative breast cancers (TNBCs), but EGFR inhibitors have not been effective in treating TNBC patients. Increasing evidence supports that autophagy was related to drug resistance at present. However, the role and the mechanism of autophagy to the treatment of TNBC remain unknown. In the current study, we investigated the effect of autophagy inhibitor to gefitinib (Ge) in TNBC cells in vitro and in nude mice vivo. Our study demonstrated that inhibition of autophagy by 3-Methyladenine or bafilomycin A1 improved Ge's sensitivity to MDA-MB-231 and MDA-MB-468 cells, as evidence from stronger inhibition of cell vitality and colony formation, higher level of G0/G1 arrest and DNA damage, and these effects were verified in nude mice vivo. Our data showed that the mitochondrial-dependent apoptosis pathway was activated in favor of promoting apoptosis in the therapy of Ge combined autophagy inhibitor, as the elevation of BAX/Bcl-2, Cytochrome C, and CASP3. These results demonstrated that targeting autophagy should be considered as an effective therapeutic strategy to enhance the sensitivity of EGFR inhibitors on TNBC.

Topics: Adenine; Animals; Autophagy; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Synergism; Female; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Macrolides; Mice; Mice, Nude; Mitochondria; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

To investigate the effect of combined administration of autophagy inhibitor 3-methyladenine/bafilomycin A1 and EGFR inhibitor gefitinib on triple-negative breast cancer MDA-MB-468, MDA-MB-231 cells and estrogen receptor-positive MCF-7 cells.. All the cells were treated with 3-methyladenine/bafilomycin A1 and/or gefitinib. The effect of autophagy inhibitor and gefitinib on the cell growth was evaluated by MTT assay. Cell apoptosis was detected by flow cytometry. Western blot analysis was used to determine the alteration of autophagy-related protein (such as LC3) and apoptosis-related proteins (such as caspase-3 and caspase-9).. MTT assay showed that the IC50 in the GE+ 3-MA and GE+ BAF groups were (4.1±0.2) μmol/L and (3.8±0.3) μmol/L, significantly lower than that of the gefitinib alone group [(7.0±0.2) μmol/L] in MDA-MB-468 cells (P<0.05). Similarly, the IC50 in the GE+ 3-MA and GE+ BAF groups were (9.7±0.1) μmol/L and (7.7±0.2) μmol/L, significantly lower than that of the gefitinib alone group [(14.7±0.1) μmol/L]in MDA-MB231 cells (P<0.05). The flow cytometry assay revealed that the apoptosis rates of MDA-MB-468 cells in GE, GE+ 3-MA and GE+ BAF groups were (12.43±3.18)%, (23.37±2.71)% and (18.71±2.81)%, respectively. The apoptosis rates of MDA-MB-231 cells of the GE, GE+ 3-MA and GE+ BAF groups were (12.15±1.82)%, (16.94±2.19)% and (33.83±5.92) %, significantly higher than that of the gefitinib alone group (All P<0.05). The apoptosis rates of the MCF-7 cells were not changed significantly among the three groups (P>0.05). Western blot data showed that the expression levels of LC3 and p-Akt were decreased in the combined groups than that of the gefitinib alone group, while the p-PTEN, caspase-3 and caspase-9 were increased.. Autophagy inhibitor may enhance the sensitivity to gefitinib in MDA-MB-468 and MDA-MB-231 cells by activation of the PTEN/P13K/Akt pathway. Apoptosis in MDA-MB-468 and MDA-MB-231 cells might be enhanced by the combination treatment through caspase cascade.

Topics: Adenine; Antineoplastic Agents; Autophagy; Autophagy-Related Proteins; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Gefitinib; Humans; Macrolides; MCF-7 Cells; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Quinazolines; Triple Negative Breast Neoplasms